the Prevalence of Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus in Processed Food samples in Riyadh, saudi Arabia

Staphylococcus aureus mainly Methicillin Resistant Staphylococcus aureus(MRsA) is a life-threatening infection that occurring in food and caused a public health concern. This study designed to examine the prevalence of S. aureus and MRSA in different types of processed food. Food samples were screened for the recovered strains of S. aureus and MRsA, and they were examined for antimicrobial susceptibility and by molecular characterization of mecA and staphylococcal cassette chromosome mec(SCCmec). Detection of virulence factors like Panton-Valentine Leukocidin (PVL), Staphylococcus aureus protein A(spa) and Staphylococcal enterotoxins(SEs) by PCR using specific primers. Among the 150 collected processed food samples, 62.7% were contaminated by S. aureus bacteria, 56.4% of which were proved as MRSA. 17% of MRSA isolates were positive for mecA genes with the SCCmec type IVb and V (11.1% each) as the solely existing types of SCCmec. None of the MRsA isolates carried mecC or mecB genes. Most of MRSA isolates were multidrug resistance and 33.3% of MRsA-mecA positive isolates also carried vancomycin resistance genes (i.e., vanB). In addition, spa gene was found among 7.5% of MRSA isolates; none of which were positive for PVL gene. Further, there were variant presence of SEs among MRSA isolates and the highest presence was from type SEH (49.1%). Generally, our results confirmed that processed foods in Saudi Arabia (Riyadh) are potential vehicles for multidrug resistant S. aureus and MRSA transmission; which are serious public health risks, and underlined the need for good hygiene practices.

LA-MRSA can be occurred by direct contact with animals or utilization of animal products e.g., lamb, calve, and goat. Yet, the spread rate of S. aureus was lower in goat and calve (12.5% and 1.4%, respectively) than it in lamb (29.7%). LA-MRSA was discovered firstly in France and in the Netherlands then it was found in the pig population in countless countries of Europe. Further, as CA-MRSA and HA-MRSA, LA-MRSA clones are different between countries; e.g., clonal complex 398 (CC398) was detected in Europe and USA, whereas clonal complex 9 (CC9) was found in Asia 3,6,11 .
However, MRSA infections spread vigorously throughout Saudi Arabia and reached 50% during 2011 in King Fahad Medical City in Riyadh city 12 . The number of studies about MRSA infection in Saudi Arabia is insufficient compared to the other countries around the world; although, MRSA in Saudi Arabia has been discovered in 1990s 13 . In spite of focusing on investigating and discussing HA-MRSA mainly, there is still a shortage in the number of studies on HA-MRSA in children and surprisingly it is found in males more than females 13 . Based on the prevalence of CA-MRSA disease, the quantity of patients significantly raised in Saudi Arabia from 9.9 per 10,000 people in 2001 to 67 per 10,000 people in 2008 13,14 . Furthermore, during the time between 2000 to 2008, the spread rate of CA-MRSA along Eastern region of Riyadh city increased by six times 12 . Also, CA-MRSA samples collected from Qatif city, Saudi Arabia showed that CA-MRSA cases increased gradually from 23% in 2006 to 60% in 2015. In terms of LA-MRSA infections, there is still a lack of studies that investigate this type of MRSA 15,16 . The purpose of this study was to investigate the prevalence of two types of MRSA: CA-MRSA and LA-MRS in processed foods collected from the capital city of Saudi Arabia and their antibiotic resistance and molecular characteristics of each isolate based on the identification of MecA gene, SCCmec types, PVL, spa gene, and SEs by PCR technique.

Sample collection
A total of 150 samples of processed foods were collected during a period of August 2019 to March 2020 from different farms and local retail markets throughout Riyadh city, Kingdom of Saudi Arabia, which are namely: A, B, C, D, E, F, G, H, and I. Samples were smoked Turkey (N=50), sausages (N=50), and salami (N=50).

Bacterial Growth Conditions
After cutting processed foods samples into small pieces by sterilized scalpel and forceps, 0.5g of each were added to approximately 5ml of specific liquid nutrient medium (i.e., brain heart infusion [BHI]) that were later incubated for 24 hours at 37°C in order to cultivate the bacteria.

Isolation and identification of S. aureus
In order to examine S. aureus colonies phenotypically, a loop of each cultivated sample was streaked on the Mannitol salt agar (MSA) -as a selective media to differentiate and isolate such bacteria-which were incubated for overnight at 37°C. As a confirmation stage, DNAs test was also performed for all S. aureus isolates. To detect the growth of MRSA isolates, we assessed the performance of MRSA chromagar as compared to that of mannitol salt agar supplemented with oxacillin (MSAO) for the recovery of MRSA from food specimens.

DNA extraction
Two colonies on MRSA chromagar plates were picked and used to be inoculated in 5 ml of BHI and grown overnight at 37°C. DNA was isolated and purified using a QIAamp DNA mini kit according to the manufacturers' instructions.
Detection of mecA, mecB , mecC and SCCmec typing of the mecA positive MRSA isolates mecA, mecB, and mecC (also named mecALGA251) were identified by PCR using primers defined previously 17,18 The mecA-positive MRSA isolates were also illustrated by SCCmec type using PCR using primers defined previously 19 .

Prevalence of S. aureus and MRSA infection in food
From 150 food samples 94 samples (62.7%) were contaminated by S. aureus bacteria. The contaminated samples were from turkey (27.7%), salami (42.6%), and sausages (29.8%). Despite the fact that S. aureus bacteria were detected in raw sausages samples, two samples were cooked for two different period of times (5 and 10 minutes) and they were still positive for S. aureus and MRSA infection as well. From the total of the 94 S. aureus isolates from different types of processed food, 53 (56.4%) isolates were confirmed as MRSA. Of the 53 MRSA isolates, 3/53 (5.7%) were from Turkey, 19/53(35.8%) were from Salami, and 31/53 (58.5%) were from Sausages (Table 1). In addition, mecA, mecB and mecC gene were examined in all subjected methicillin resistant S. aureus (MRSA) isolates. The result indicated that 17% of MRSA isolates (9/53) were positive for mecA genes, while none of them were positive for mecC or mecB genes. Antibiotic resistance profiles of MRSA isolates A total of 8 antimicrobial agents were examined for antimicrobial susceptibility in all MRSA isolates. Hence, the percentages of the bacterial resistance for each one of these antibiotics from all kinds of the examined samples are: 30.2% (16/53) to penicillin, 20.8% (11/53) to ampicillin, 86.8% (46/53) to oxacillin, 34% (18/53) to erythromycin, 9.4% (5/53) to gentamicin, 24.5% (13/53) to oxytetracycline, 47.2% (25/53) to trimethoprim-sulphamethoxazole, and 22.6% (12/53) to vancomycin. As regards to vancomycin resistance genes (i.e., vanA and vanB), vancomycin resistant isolates were tested and found that 33.3% (4/12) of them carried vanB gene but none of them were carried vanA gene and their origins were from salami (2), and sausages (2). Noticeably, oxacillin was the most commonly detected antibiotic among sausage samples, while trimethoprim-sulphamethoxazole resistance was detected at lower frequencies among turkey samples in MRSA isolates in comparison with other types of food isolates (i.e., salami origin) ( Fig. 1 and Table 2). However, most of MRSA isolates were resistant to more than three antibiotics. For instances, 13.2% (7/53) of such isolates were resistant to 5-7 antibiotics (five from salami, and two from sausages), 43.4% (23/53) of them were resistant to 2-4 antibiotics (16 from sausages, six from salami, and one from turkey origin) and 5.7% (3/53) of them were resistant to all eight antibiotics (one from salami, and two from sausages). On the other hand, 64.2% (34/53) of the isolates were sensitive to 5-7 antibiotics, 11.3% (6/53) of them were sensitive to 2-4 antibiotics, and 7.5% (4/53) of them were sensitive to the all eight antibiotics.

Molecular typing of MRsA isolates
The positive mecA isolates were exposed to identify five different types of SCCmec: type I, II, III, IV, and V. consequently, SCCmec types between seven out of nine isolates that carried mecA gene (from salami and sausages origins) could not be detected, whereas only two isolates had a single type which are SCCmec type V and IVb and they were from sausages origin, Table 3.

Distribution of virulence genes
In the tested MRSA samples (53 isolates), Staphylococcus aureus protein A (spa) gene was found between 4 out of 53 (7.5%) and none of them were positive to the Panton Valentine leukocidin (PVL) gene.  27 ), there were variant presence of these types among such isolates. With regard to the presence of 14 investigated enterotoxin genes between examined samples, the highest proportion was from SEH (49.1%) followed by SEM (11.3%), SEA and SEN (7.5% each), SEO and SEK (5.7% each), SEE (3.8%), and then SEB, SEG, and SEI (1.9% each). Whilst, none of SEC, SED, SEJ, and SEL were existed in any of MRSA isolates (Fig. 2). Overall, MRSA isolates that were derived from turkey samples did not carry any of the 14 investigated enterotoxin genes. Staphylococcal enterotoxin (SEA, SEB, SEC, SED, and SEE) and their origins were from salami (4/6 [66.7%]) and sausages (2/6 [33.3%]) samples. The majority of classic staphylococcal enterotoxins SCCmec= staphylococcal cassette chromosome mec and their origins were from salami and sausages but the highest presence of egc was from sausages samples (i.e., 66.7%). Moreover, some described combinations of the virulence genes of S. aureus were spotted. The SEC-SEL gene combination, typical of the SaPIbov pathogenicity island, was not detected in any of the isolates, as shown in Fig. 2 and Table 4.

disCussiON
Because of CA-MRSA is reported to be less serious outcomes than HA-MRSA, HA-MRSA was investigated more than CA-MRSA and LA-MRSA locally 16,28 . Apart from samples origins, S. aureus has been known to be an important reason for causing animal illnesses like LA-MRSA (which can transmit to humans via livestock over handling, close contact, or animal-derived food consumption) and food poisoning; especially when such bacteria are multidrug resistant and have the ability to produce toxins like enterotoxins 11,[29][30][31][32] .
Hence, turkey, salami, and sausages have been checked for the first time for S. aureus and MRSA in this study; which were collected from different huge supermarkets around Riyadh city. This current study has found approximately 62.7% recorded presence of S. aureus between the tested samples and it was significantly higher than it (18%) in Islam et al. (2019) study in Dhaka, Bangladesh 29 . Further, more than half of such contaminated samples (56.4%) from processed food considered to be CA-MRSA infection and it occurred through the food preparing process or even hygienic handling circumstances, according to Kluytmans (2010) 33 . Moreover, turkey samples recorded as the lowest percentage of CA-MRSA (5.7%) followed by salami (35.8%) then sausages (58.5%) from all different collecting places. This may be due to the lower content of fat comparing with the rest of our processed food samples, considering to be rich nutrient environment for bacterial growth, as what confirmed by Raji et al. (2016) when poultry retail meat from Riyadh city were examined 16 . MecA gene that is responsible for the resistance to methicillin and located on SCCmec was discovered for the first time in Riyadh city among processed food. In fact, MecA gene was found between 9 out of 53 of MRSA isolates (17%) and the ratios of which were diverse between samples (i.e., 55.6% from salami and 44.4% from sausages samples). In addition, our study found that about 86.8% of the positive mecA isolates were resistant to Oxacillin which was confirmed by Osman et al. (2017) about the negative mecA strains are resistant to Oxacillin rather than methicillin or could be a novel of mecA homologue that was linked with cattle 35 . Moreover, a study was done by Elhaaan et al, stated that it is extremely suggested to consider alternate mechanisms for β-lactams resistance that may compete with mecA  36 .

Antibiotic resistance profiles of MRSA isolates
The susceptibility test of a fifty-three MRSA isolates in this study was estimated for eight antibiotics from different classes and the resistance was detected against Oxacillin (86.8%), which was higher comparing with Gentamicin (9.4 %). It is worth noting that 13.2% of MRSA isolates were sensitive to Oxacillin. This result confirmed the study of Saeed et al. (2014) who stated that Methicillin resistant Staphylococcus aureus (MRSA) is phenotypically presenting minimum inhibitory concentration (MIC) of oxacillin greater than 2 mg/L. Nevertheless, lately, cefoxitin/oxacillinsusceptible mecA-positive S. aureus (OS-MRSA) has been described globally 37 .
Such giant resistance; in addition, was stated by Osman et al. (2017) to the over using of antibiotics in order to promote the animal growth or treatment 35 . The widespread of multidrug resistant phenotypes among MRSA isolates around Saudi Arabia generally was accounted by Adam and Abomughaid (2018) for the significant number of pilgrims whom may carry these such drug resistant bacteria 38 . Surprisingly, this study uncovered 22.6% (12/53) were resistant to Vancomycin and 33.3% (4/12) were carried vanB gene (from salami [2], and sausages [2]); which is still using as a drug of choice to cure MRSA infections, according to Gade and Qazi (2013) 39 .
The mec A carrier (i.e., SCCmec) type I, II, III, IV, and V were also screened in this study among the positive mec A samples in order to achieve the goal of the epidemiology study for the MRSA isolates. It was found two types among two samples from salami and sausage (types IVb and V). In addition, this study completely support the studies done by Deurenberg 27,43 .
Positive MRSA isolates in our study found to be harbored one or more genes from classic staphylococcal enterotoxins (SEA, SEB, SEC, SED, and SEE) and the enterotoxin gene cluster (egc [SEG, SEI, SEN, SEO, and SEM]). In addition, our result confirms that SEH (49.1%) was the most spread enterotoxin followed by SEM (11.3%), 7.5% from each one of SEA and SEN, 5.7% from each one of SEO and SEK, SEE (3.8%), and 1.9% from each one of SEB, SEG, and SEI.

CONClusiON
From the above, this is the first comprehensive investigation of the prevalence of S. aureus and MRSA in prossesed foods from Riyadh city, Saudi Arabia. This study declared a relatively high ratios of S.aureus and MRSA contamination in such foods (62.7% and 56.4%, respectively). Importantly, two cooked sausages samples were still contaminated with such bacteria and infection and none of MRSA isolates were positive for PVL gene. Whilst, nine of MRSA isolates were positive for MecA gene and spa gene was among four of which. High amount of antimicrobial resistance also found between the isolates ,therefore, our data approved the possible role of processed foods in the transmission of multidrug resistant S.aureus strains and successful MRSA in Riyadh , Saudi Arabia.

ACkNowLEDGMENTS
My thanks must go to Princess Nourah bint Abdulrahman University for its financial support as well as the Health Sciences Research Center/core facilities for allowing me to complete the analysis of my research samples in their cutting-edge laboratory.